Lysine acylation is a ubiquitous post-translational modification (PTM) that plays pivotal roles in various cellular processes, such as transcription, metabolism, protein location and folding. Thousands of lysine acylation sites have been identified based on advances in antibody enrichment strategies, highly sensitive mass spectrometry (MS) analysis and bioinformatics. However, only 27 lysine methacrylation (Kmea) sites were identified, and they are exclusively located on histones. It is hard to separate, purify and differentiate Kmea peptides or proteins from its structural isomer lysine crotonylation (Kcr) with general biochemical approaches. Here, for the first time, we identified Kmea sites on a non-histone protein, Cyclophillin A (CypA). To investigate the function of Kmea on CypA, we developed a general genetic code expansion approach to incorporate unnatural amino acid (Uaa) ε-N-Methacryllysine (MeaK) into target proteins and identified interacting proteins of methacrylated CypA using affinity-purification MS.